CD95L (also known as FasL) is a transmembrane ligand that binds to the so-called death receptor CD95 (also known as Fas/APO1). Metalloproteases can cleave CD95L to produce the soluble ligand cl-CD95L. While transmembrane CD95L is a potent apoptosis inducer that participates in immune surveillance and tolerance (see review32), cl-CD95L cannot induce apoptosis, but instead triggers the motility of cancer cells and activated T lymphocytes12, 15, 34 and aggravates inflammation23, 34. Binding of transmembrane CD95L to CD95 leads to formation of the death-inducing signaling complex (DISC), which contains the Fas-associated death domain adaptor protein and caspases −8 and −1014. This complex plays a pivotal role in the initiation of apoptotic signaling. Cells can be classified as type I or type II according to the efficiency with which DISC forms and their reliance on the mitochondrion-driven apoptotic signal to implement the CD95-mediated apoptotic signal14. It was previously shown that type II cells are of epithelial lineage, whereas type I cells are mesenchymal-like1. Type II cells are more sensitive to the CD95-mediated apoptotic signal than type I cells; therefore, it was concluded that cancer cells undergoing epithelial-to-mesenchymal transition (EMT) reprogram their apoptotic machinery to resist CD95-mediated cell death 1. These findings encouraged the inventors to investigate whether EMT alters the cellular response to cl-CD95L, i.e., motility.
Sphingolipids (SLs) belong to a class of lipids whose members play multiple roles in eukaryotic cells. SLs are important structural components of membranes and are second messengers that regulate cell growth, differentiation, and death10. Ceramide (N-acyl-D-erythro-sphingosine) can be generated via hydrolysis of sphingomyelin by sphingomyelinases, or via ceramide synthase (CerS)-mediated de novo ceramide biosynthesis. This pathway involves acylation of sphinganine with fatty acyl-CoAs of chain lengths of C14-C26 to produce dihydroceramide. Alternatively, CerS can synthesize ceramide by the salvage pathway through direct acylation of sphingosine, which is derived from SL catabolism10. Ceramide synthesis is orchestrated by six mammalian CerS proteins, each of which produces ceramides with restricted acyl chain lengths20. Among these proteins, ceramide synthase isoform 6 (CerS6) was most recently cloned37. Although most studies on ceramides have analyzed their impact on apoptotic signaling, these lipids also promote the formation of ordered liquid phases called lipid rafts, whose accumulation rigidifies the plasma membrane28. Ceramides form rigid ceramide-enriched domains when their N-acyl chain is longer than C125, 7 and lead to an overall decrease in ordered lipid membranes in CerS2-null mice29. The biophysical properties of hepatocyte membranes are modified in CerS2-null mice, including increased fluidity24, which is directly associated with changes in the SL composition. Furthermore, high plasma membrane fluidity is correlated with enhanced migratory/invasive ability of tumor cells22, 30, 33, 39, which might be due to the enhanced deformability of these cells22. These observations raise the possibility that EMT alters the level of SL biosynthesis to increase membrane fluidity, which in turn promotes cell migration, a phenotypic change associated with EMT.